Introducing the Peroxicats consortium
Each partner has been selected and invited to join the PEROXICATS consortium
based on its excellence in the different S&T areas needed to build up this challenging interdisciplinary project.
Peroxicats consortium at the kick-off meeting in Madrid.
In this way, the four research/academic groups (one small but highly dynamic University and three large research Institutes) and the two industrial partners (a world-leader company and a highly-specialized SME) have the expertises required in the fields of:
Fungal Biology and Ecology
Biochemistry and Molecular Biology of Peroxidases
Enzyme Structure-function and Engineering
Fernandez-Fueyo E, Ruiz-Dueñas FJ, Martínez AT (2014). "Engineering a fungal peroxidase that degrades lignin at very acidic pH" Biotechnology for Biofuels, 7. 114.
Fernandez-Fueyo E, Ruiz-Dueñas FJ, Martínez AT
Engineering a fungal peroxidase that degrades lignin at very acidic pHBiotechnology for Biofuels, 7. 114.
Ligninolytic peroxidases are divided into three families: manganese peroxidases (MnPs), lignin peroxidases (LiPs), and versatile peroxidases (VPs). The latter two are able to degrade intact lignins, as shown using nonphenolic lignin model compounds, with VP oxidizing the widest range of recalcitrant substrates. One of the main limiting issues for the use of these two enzymes in lignocellulose biorefineries (for delignification and production of cellulose-based products or modification of industrial lignins to added-value products) is their progressive inactivation under acidic pH conditions, where they exhibit the highest oxidative activities.Results
In the screening of peroxidases from basidiomycete genomes, one MnP from Ceriporiopsis subvermispora
was found to have a remarkable acidic stability. The crystal structure of this enzyme recently became available and, after comparison with Pleurotus ostreatus
VP and Phanerochaete chrysosporium
LiP structures, it was used as a robust scaffold to engineer a stable VP by introducing an exposed catalytic tryptophan, with different protein environments. The variants obtained largely maintain the acidic stability and strong Mn2+-oxidizing activity of the parent enzyme, and the ability to oxidize veratryl alcohol and Reactive Black 5 (two simple VP substrates) was introduced. The engineered peroxidases present more acidic optimal pH than the best VP from P. ostreatus
, enabling higher catalytic efficiency oxidizing lignins, by lowering the reaction pH, as shown using a nonphenolic model dimer.Conclusions
A peroxidase that degrades lignin at very acidic pH could be obtained by engineering an exposed catalytic site, able to oxidize the bulky and recalcitrant lignin polymers, in a different peroxidase type selected because of its high stability at acidic pH. The potential of this type of engineered peroxidases as industrial biocatalysts in lignocellulose biorefineries is strongly enhanced by the possibility to perform the delignification (or lignin modification) reactions under extremely acidic pH conditions (below pH 2), resulting in enhanced oxidative power of the enzymes.
External web link
Kellner H, Luis P, Pecyna MJ, Barbi F, Kapturska D, Krüger D, Zak DR, Marmeisse R, Vandenbol M, Hofrichter M (2014). "Widespread Occurrence of Expressed Fungal Secretory Peroxidases in Forest Soils" PlosOne, 9.
Kellner H, Luis P, Pecyna MJ, Barbi F, Kapturska D, Krüger D, Zak DR, Marmeisse R, Vandenbol M, Hofrichter M
Widespread Occurrence of Expressed Fungal Secretory Peroxidases in Forest SoilsPlosOne, 9.
Fungal secretory peroxidases mediate fundamental ecological functions in the conversion and degradation of plant biomass. Many of these enzymes have strong oxidizing activities towards aromatic compounds and are involved in the degradation of plant cell wall (lignin) and humus. They comprise three major groups: class II peroxidases (including lignin peroxidase, manganese peroxidase, versatile peroxidase and generic peroxidase), dye-decolorizing peroxidases, and heme-thiolate peroxidases (e.g. unspecific/aromatic peroxygenase, chloroperoxidase). Here, we have repeatedly observed a widespread expression of all major peroxidase groups in leaf and needle litter across a range of forest ecosystems (e.g. Fagus
, and Populus
spp.), which are widespread in Europe and North America. Manganese peroxidases and unspecific peroxygenases were found expressed in all nine investigated forest sites, and dye-decolorizing peroxidases were observed in five of the nine sites, thereby indicating biological significance of these enzymes for fungal physiology and ecosystem processes. Transcripts of selected secretory peroxidase genes were also analyzed in pure cultures of several litter-decomposing species and other fungi. Using this information, we were able to match, in environmental litter samples, two manganese peroxidase sequences to Mycena galopus
and Mycena epipterygia
and one unspecific peroxygenase transcript to Mycena galopus
, suggesting an important role of this litter- and coarse woody debris-dwelling genus in the disintegration and transformation of litter aromatics and organic matter formation.
External web link
Molina-Espeja P, García-Ruiz E, González-Pérez D, Ullrich R, Hofrichter M, Alcalde M (2014). "Directed evolution of Unspecific Peroxygenase from Agrocybe aegerita" Appl. Environ. Microbiol., doi: 10.1128/AEM.00490-14.
Molina-Espeja P, García-Ruiz E, González-Pérez D, Ullrich R, Hofrichter M, Alcalde M
Directed evolution of Unspecific Peroxygenase from Agrocybe aegeritaAppl. Environ. Microbiol., doi: 10.1128/AEM.00490-14.
Unspecific peroxygenase (UPO) represents a new type of heme-thiolate enzyme with self-sufficient mono(per)oxygenase activity and many potential applications in organic synthesis. With a view to taking advantage of these properties, we subjected the Agrocybe aegerita UPO1 encoding gene to directed evolution in Saccharomyces cerevisiae. To promote functional expression, several different signal peptides were fused to the mature protein and the resulting products tested. Over 9,000 clones were screened using an ad-hoc dual-colorimetric assay that assessed both peroxidative and oxygen-transfer activities. After 5 generations of directed evolution combined with hybrid approaches, 9 mutations were introduced that resulted in a 3,250-fold total activity improvement with no alteration in protein stability. A breakdown between secretion and catalytic activity was performed by replacing the native signal peptide of the original parental type with that of the evolved mutant: the evolved leader increased functional expression 27-fold whereas a 18-fold improvement in kcat/Km for oxygen transfer activity was obtained. The evolved UPO1 was active and highly stable in the presence of organic co-solvents. Mutations in the hydrophobic core of the signal peptide contributed to enhance functional expression up to 8 mg/L, while catalytic efficiencies for peroxidative and oxygen transfer reactions were increased by several mutations in the vicinity of the heme-access channel. Overall, the directed evolution platform described is a valuable point of departure for the development of customized UPOs with improved features and for the study of structure-function relationships.
External web link
Hofrichter M, Ullrich R (2014). "Oxidations catalyzed by fungal peroxygenases" Curr. Opin. Chem. Biol., 19. 116-125.
Hofrichter M, Ullrich R
Oxidations catalyzed by fungal peroxygenasesCurr. Opin. Chem. Biol., 19. 116-125.
The enzymatic oxyfunctionalization of organic molecules under physiological conditions has attracted keen interest from the chemical community. Unspecific peroxygenases (EC 126.96.36.199) secreted by fungi represent an intriguing enzyme type that selectively transfers peroxide-borne oxygen with high efficiency to diverse substrates including unactivated hydrocarbons. They are glycosylated heme-thiolate enzymes that form a separate superfamily of heme proteins. Among the catalyzed reactions are hydroxylations, epoxidations, dealkylations, oxidations of organic hetero atoms and inorganic halides as well as one-electron oxidations. The substrate spectrum of fungal peroxygenases and the product patterns show similarities both to cytochrome P450 monooxygenases and classic heme peroxidases. Given that selective oxyfunctionalizations are among the most difficult to realize chemical reactions and that respectively transformed molecules are of general importance in organic and pharmaceutical syntheses, it will be worth developing peroxygenase biocatalysts for industrial applications.
External web link
"Search, engineering, and applications of new oxidative biocatalysts"
Biofuels, Bioproducts & Biorefining (2014)
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